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Structure and Function of Sodium-Calcium Exchangers

钠钙交换体的结构和功能

基本信息

批准号：
8841747
负责人：
Dinesh A Yernool
金额：
$ 28.67万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-15 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8841747
关键词：
Active Biological Transport Architecture Binding Sites Biochemical Calcium Calcium ion Cardiac Cardiac Muscle Contraction Cardiac Myocytes Cations Cell Death Cell division Cell membrane Cell physiology Cells Chimeric Proteins Communities Complex Crystallization Data Development Disease Drug Design Elements Family Fertilization Foundations Future Genetic study Goals Health Heart Diseases Homeostasis Homologous Gene Ii-Key Immunoglobulin Fragments Integral Membrane Protein Ion Channel Ion Transport Kinetics Knowledge Laboratories Lead Life Liposomes Membrane Membrane Proteins Membrane Transport Proteins Methodology Methods Mission Modeling Molecular Molecular Genetics Myocardial Ischemia Myocardial dysfunction Neurons Outcome Physiological Property Protein Family Proteins Proteolysis Public Health Pump Recurrence Regulation Research Resolution Signal Transduction Sodium-Calcium Exchanger Specificity Structural Models Structure Structure-Activity Relationship Study models Time Toxic effect Transmembrane Domain United States National Institutes of Health X-Ray Crystallography base biophysical properties cost design experience improved inhibitor/antagonist neuron loss neurotransmission novel novel strategies optical imaging overexpression receptor reconstitution small molecule structural biology three dimensional structure three-dimensional modeling transport inhibitor

项目摘要

DESCRIPTION (provided by applicant): Flux of calcium ions, potent signals, controls many if not all cellular processes from fertilization and cell division to eventual cell death. Sodium/calcium exchangers (NCX), high-capacity integral membrane transporters, are the principal Ca2+ pumps in the plasma membranes of most cells. The unmatched ability of NCX to rapidly extrude Ca2+ using preexisting Na+-gradient potentials is critical to the normal function of excitable cells such as cardiac myocytes and neurons. These cells undergo quick, recurrent oscillations in levels of intracellular Ca2+ to achieve cardiac muscle contraction and neuronal signaling, respectively; and perturbations in NCX activity have been implicated in cardiac dysfunction and neuronal cell death. At present, an atomic-resolution structure of NCX has not been determined. Lack of 3D structure impedes the elucidation of structure-function relationships that are key to understanding NCX's mechanism of action. Our long-term goal is to describe at the atomic level the mechanism of transport and regulation of Ca2+/cation exchange in cardiac and neuronal cells. Toward this goal, the specific aims proposed here are as follows: 1. Heterologous overexpression of membrane proteins using a novel method. Optimized expression methods developed in my laboratory to increase membrane protein yields and reduce their host toxicity will enable us to produce enough protein for biochemical, biophysical, and crystallographic analyses. Initial expression targets will be prokaryotic NCX homologs selected for their significant sequence identity to eukaryotic sodium-calcium exchangers. Subsequently we will demonstrate our method's applicability to a large panel of membrane protein targets and investigate the molecular basis of overexpression toxicity. 2. Demonstration that prokaryotic NCX is an appropriate model for studies of Na+/Ca2+ exchange. This will be achieved by determining the oligomerization state and topology of the transporter, followed by characterization of structure-function and transport properties of prokaryotic NCX homologs by reconstitution into liposomes. Wild-type ion transport kinetics will be determined and inhibitors of transport identified. 3. Structure. We will crystallize a prokaryotic NCX homolog(s) alone and/or in complex with antibody fragments to determine and analyze structure(s). Combining biochemical and biophysical data with the crystal structure of an NCX homolog will lead to development of structure-based mechanistic models for Na+-Ca2+ exchange, spur new structure-based hypotheses to understand the mechanism of cardiac NCX, and may inspire structure-based drug design to find pharmacologically active agents.

描述（由申请人提供）：钙离子的通量是一种强有力的信号，控制着从受精、细胞分裂到最终细胞死亡的许多（如果不是全部）细胞过程。钠/钙交换器（NCX）是一种高容量的整体膜转运体，是大多数细胞质膜中主要的Ca2+泵。NCX利用预先存在的Na+梯度电位快速挤出Ca2+的无与伦比的能力对可兴奋细胞（如心肌细胞和神经元）的正常功能至关重要。这些细胞经历细胞内Ca2+水平的快速、反复振荡，分别实现心肌收缩和神经元信号传导；NCX活性的紊乱与心功能障碍和神经元细胞死亡有关。目前，一个原子分辨率